Systemic and Phloem-Specific Protein Targeting by High Affinity Nanobodies Expressed From a Plant RNA Virus Vector

Area of Science:

• Plant biotechnology
• Molecular biology
• Virology

Background:

• Nanobodies (Nbs) show promise for plant biotechnology but face challenges in expression and targeting.
• Current methods for in planta Nb expression are time-consuming or lack systemic reach.
• Tissue-specific expression of Nbs has not been previously studied.

Purpose of the Study:

• To establish a proof-of-concept platform for phloem-specific protein targeting using nanobodies.
• To demonstrate the utility of a viral vector for Nb expression in plant phloem.
• To validate Nb-antigen interactions within the phloem using advanced imaging techniques.

Main Methods:

• Utilized a citrus tristeza virus-based vector for nanobody expression.
• Generated transgenic Nicotiana benthamiana plants expressing GFP-tagged proteins.
• Employed pull-down assays and fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) for interaction validation.

Main Results:

• Successfully expressed anti-green fluorescent protein (GFP) nanobodies specifically in the phloem.
• Confirmed interaction between anti-GFP nanobodies and GFP-tagged proteins/peptides.
• Validated Nb-antigen interactions in the phloem using FRET-FLIM, a novel application.

Conclusions:

• The developed viral vector platform enables efficient, phloem-specific nanobody expression.
• This approach is valuable for studying nanobody-mediated functions in plant phloem.
• The platform facilitates targeting and identification of phloem proteins and virus-interacting factors.